NADPH oxidase (NOX) appears to play a role in the development of myocardial hypertrophy and subsequent progression of heart failure. However, the signaling mechanisms of NOX in cardiac myocytes is unknown. The long-term goal of this research is to understand the signaling mechnisms of NOX in the development of myocardial hypertrophy and heart failure induced by excessive adrenergic stimulation and hemodynamic pressure overload. Our prelminary data indicate that p67phox, the major cytosolic subunit of NOX, mediates the development of myocardial hypertrophy through a novel reactive oxygen species (ROS) - independent mechanism. Based on these data, we hypothesize that NOX/p67phox mediates myocardial hypertrophy that is induced by hypertrophic stimuli including 11-AR-stimulation and hemodynamic pressure overload, and its signaling function involve a novel ROS-independent mechanism. To test this hypothesis, we proposed the following three Specific Aims: In Specific Aim 1, we will determine whether NOX/p67phox mediates 11-adrenergic receptor (11-AR)- stimulated myocardial hypertrophy and whether this involves a novel ROS-independent signaling mechanism in vitro. Dominant negative (DN) mutants and wild type (WT) contstructs of two NOX cytosolic subunits, p67phox and p47phox. Mutants or WT constructs will be overexpressed in cultured adult rat ventricular myocytes (ARVM). The effects of these mutants or WT constructs on 11-AR stimulation-induced hypertrophy and NOX enzyme activity will be determined. The potential protein-protein interaction between p67phox and MEK-ERK cascade will also be determined in this aim. In Specific Aim 2, we will test if p67phox mediates 11-AR-stimulated cardiac hypertrophy in vivo. We will use the newly generated heterozygous DN-p67 transgenic (TG) mice with cardiac-specific overexpression of the DN p67phox mutant to generate homozygous DN-p67 TG mice. Since the commercially available TG mice with cardiac-specific overexpression of a constitutively activated mutant of 11B-AR (CAM-11B-AR) develop cardiac hypertrophy, we will crossbreed these two TG lines (DN-p67 and CAM-11B-AR) to study the effect of overexperssion of DN p67phox mutant protein in the heart on 11B-AR-induced myocardial hypertrophy in vivo. In Specific Aim 3, we will test the role of myocardial p67phox in mediating hemodynamic overload- induced myocardial hypertrophy in vivo. The homozygous DN-p67 TG mice and age- and gender-matched WT control mice will be subjected to chronic pressure overload caused by ascending aorta constriction (AAC). The cardiac phenotypes and function of these TG and WT mice with AAC or sham operation will be studied to determine the role of p67phox in mediating chronic pressure overload-induced myocardial hypertrophy in vivo. Our studies will provide new understanding of the role and novel mechanisms of NOX/p67phox in the development of cardiac hypertrophy that should lead to novel treatments for congestive heart failure.